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T Osawa

Publications and source records attributed to T Osawa.

At least 235 records · Page 13Linked to original sources
PubMed1990

In vivo localization of antitumor lymphocytes separated with Dolichos biflorus lectin.

We examined the in vivo localization of Con A sup-cultured murine splenocytes separated with Dolichos biflorus agglutin (DBA) to elucidate the mechanism underlying the high antitumor efficacy of cells which are not agglutinable with DBA (DBA- cells). First, 51Cr-labeled DBA-separated cells were injected intravenously (i.v.) into mice and then the tissues were collected for gamma counting. DBA- cells showed significant localization in the spleen, compared with DBA+ cells. This was further confirmed by injecting Thy1.2-positive DBA- cells into Thy1.1 congenic mice. Furthermore, after [3H]TdR-labeled DBA-separated cells had been injected i.v., the spleens of the recipients were fixed, sectioned and processed for autoradiography to determine in which region of the spleen the labeled cells had accumulated. We found that DBA- cells were especially concentrated in the white pulp of the spleen, where lymphocytes are localized before being recirculated. These results suggest that DBA- cells exert strong antitumor activity in vivo partly due to their localization in the spleen and partly to the high incidence of active recirculation.

Animals↗
PubMed1990

Anti-tumour efficacy of mouse spleen cells separated with Dolichos biflorus lectin (DBA) in experimental pulmonary metastasis of B16 melanoma cells.

Anti-tumour effector cells were generated through 4 days culture of normal C57BL/6 splenocytes in a medium containing concanavalin A supernatant and then fractionated with Dolichos biflorus lectin (DBA) into DBA+ (agglutinable with DBA) and DBA- (non-agglutinable with DBA) cells. The DBA- cells, infused intravenously into mice together with B16 melanoma cells, or adoptively transferred into mice 3 days after the injection of B16 cells, caused a marked decrease in the number of lung nodules, while the DBA+ cells exerted no effect. On the other hand, the DBA+ cells exhibited higher cytolytic activity in vitro than the DBA- cells in short-term 51Cr-release assays. Then, we analysed the mechanism of the strong anti-tumour activity of DBA- cells in vivo. We found that DBA- cells showed higher response to recombinant interleukin-2 (rIL-2) than DBA+ cells and proliferated very well with a small amount of IL-2. In addition, DBA- cells adhered more strongly to lung endothelial cells than DBA+ cells in response to rIL-1 or rTNF. Furthermore, DBA- cells produced larger amounts of macrophage activating factor (MAF) including IFN-gamma when cultured with B16 melanoma. Taken together, our results show that DBA- cells are effective in reducing experimental pulmonary metastases not only by the direct lytic activity but also by the indirect killing activity through the activated macrophage.

Animals↗
PubMed1990

Low-molecular-weight GTP-binding proteins serving as ADP-ribosylation substrate for ADP-ribosyltransferase from Clostridium botulinum and their relation to phosphoinositides metabolism in thymocytes.

ADP-ribosyltransferase from Clostridium botulinum type C strain was found to induce an increase of inositol phosphates (IPs) formation in murine thymocytes membranes. Incubation of electropermeabilized murine thymocytes with the enzyme also caused an increase of IPs formation in the cells. This increase of IPs formation in the enzyme-treated membranes and electropermeabilized cells was dependent on the amount of both NAD and the enzyme, suggesting that the stimulation of phosphoinositide-specific phospholipase C (PLC) was related to ADP-ribosylation of membrane proteins by the enzyme. On the other hand, in calf and murine thymocytes two proteins with the same molecular weight of 21,000 were found to be ADP-ribosylated by the botulinum ADP-ribosyltransferase. A minor ADP-ribosylation substrate was shown by two-dimensional polyacrylamide gel electrophoresis to be G21k, a low-molecular-weight GTP-binding protein (G protein) suggested previously by us to be involved in PLC regulation [Wang, P. et al. (1987) J. Biochem. 102, 1275-1287; (1988) 103, 137-142; and (1989) 105, 461-466], and the other major ADP-ribosylation substrate was identified as a rho A protein. Under the experimental conditions of the IPs formation study, ADP-ribosylation of both G21k and rho A proteins by botulinum ADP-ribosyltransferase in membranes and permeabilized cells was observed. These results suggest that botulinum ADP-ribosyltransferase-induced PLC stimulation in thymocytes is closely correlated with ADP-ribosylation of the low-molecular-weight G proteins.

Adenosine Diphosphate↗
PubMed1990

Heterogeneity in lectin-binding characteristics of human lymphokine-activated killer cells.

The binding of various lectins to human lymphokine-activated killer (LAK) cells was investigated. Human peripheral blood lymphocytes were cultured for 3 days with recombinant interleukin-2 (rIL-2), and then stained with 20 kinds of lectins. There were four types of lectin-binding patterns: (a) negative staining; (b) weakly positive staining; (c) strongly positive staining; and (d) two populations, one weakly and one strongly positive staining. Among the 20 kinds of lectins, both Lens culinaris agglutinin (LCA) and Pisum sativum agglutinin (PSA) showed two peaks in the staining patterns on LAK cells (type 4). We separated these two populations of LAK cells by using a cell sorter, and assayed them for cytotoxic activity against 51Cr-labeled Hela cells. The LAK cells that were strongly stained by LCA or PSA [LCA (or PSA)-bright LAK cells] showed stronger cytotoxic activity than the LAK cells that were weakly stained by LCA or PSA [LCA (or PSA)-dull LAK cells]. Furthermore, we separated lymphocytes with LCA or PSA before culturing them with rIL-2 (LAK precursor), and we found that both LCA-bright and PSA-bright lymphocytes can develop into LAK cells with higher cytolytic activity. LCA-bright lymphocytes kept their ability to be stained strongly with LCA even after culture with rIL-2 for 3 days. Similarly, LCA-dull lymphocytes were still stained weakly after culture with rIL-2. Two-color assay of LAK cells revealed that 78% of NK (CD16+)-LAK cells and 46% of T (CD3+)-LAK cells were LCA-bright cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1989

Macrophage chemotactic factor (MCF) produced by a human T cell hybridoma clone.

A human T cell hybridoma clone, D6-18, producing high levels of macrophage chemotactic factor (MCF) was established by the emetine-actinomycin D selection method. MCF was found to be present not only in the culture medium but also in the cell lysate of D6-18 cells. The secretion of the MCF from D6-18 cells was effectively inhibited by disodium cromoglycate, which is an inhibitor of the degranulation of mast cells, suggesting that MCF is stored in granules. The MCF of D6-18 cells was purified from the sonicated cell lysate by ion-exchange chromatographies and high-performance liquid chromatography. The amino acid sequence of the purified MCF was revealed to be WLGREDGSE or WLGRQDGSE. The synthetic peptide WLGREDGSE showed chemotactic activity against guinea pig macrophages and human monocytes at the concentration of about 10(-8) M.

Amino Acid Sequence↗
PubMed1989

Poly[N-acetyl-lactosamine]-type sugar chains in CD45 antigens of abnormal T cells of lpr mice are different from those of normal T cells and B cells.

The lymph node (LN) T cells from autoimmune MRL/MpJ-lpr/lpr (lpr) mice and control MRL/MpJ-+/+ (+/+) mice were compared as to their cell surface lectin binding sites and the glycoproteins responsible for the lectin binding. T cells from enlarged lymph nodes of lpr mice were found to express more binding sites for lectins which are reactive to poly[N-acetyl-lactosamine]-type sugar chains than normal +/+ mouse lymph node T cells. Furthermore, we found that high mol. wt (180,000-220,000) glycoproteins on lpr T cells were strongly stained with these poly [N-acetyl-lactosamine]-binding lectins on Western-blotting. These glycoproteins were found to belong to the CD45 family on immunoprecipitation and absorption with monoclonal anti-CD45 antibody. Thus, aberrant expression of high mol. wt CD45 (CD45R) antigens on lpr T cells may contribute greatly to the strong reaction of these cells with poly[N-acetyl-lactosamine]-binding lectins. We also found that poly[N-acetyl-lactosamine]-type sugar chains are more abundant on B cells than on lpr T cells, and that the molecular weights and the carbohydrate moieties of CD45R antigens on lpr T cells are different from those of CD45R antigens on +/+ spleen B cells.

Animals↗
PubMed1989

Immunomodulatory effects of neurotropin through the recovery of interleukin-2 production in autoimmune-prone (NZB/NZW) F1 mice.

The immunomodulatory effects of Neurotropin, a substance extracted from inflammatory skin of rabbits inoculated with vaccinia virus, were assessed in autoimmune-prone (NZB/NZW) F1 (B/W F1) mice. The concanavalin A (Con A)-induced proliferative response of spleen cells was markedly decreased in aged B/W F1 mice as compared with young B/W F1 mice. Neurotropin, when administered i.p. to aged B/W F1 mice, significantly increased the Con A-induced proliferative response. In aged B/W F1 mice, interleukin-2 (IL-2) production by Con A-stimulated spleen cells was severely impaired and IL-2 responsiveness of Con A-activated spleen cells was partially decreased in comparison with young B/W F1 mice. Neurotropin, administered to the aged B/W F1 mice, restored IL-2 production by Con A-stimulated spleen cells to the level of young B/W F1 mice. Furthermore, Neurotropin completely restored the IL-2 responsiveness of Con A-activated spleen cells from aged B/W F1 mice. To test whether Neurotropin exerts its immunoregulatory activities in B/W F1 mice by restoring IL-2 production, we directly examined the effect of recombinant IL-2 on the immune functions of spleen cells in vitro. Recombinant IL-2 markedly enhanced Con A-induced proliferative response of aged B/W F1 mice. Furthermore, the suppressive activity of spleen cells which had been activated by Con A in the presence of rIL-2 was significantly increased. These results indicate that some immunoregulatory functions of aged B/W F1 mice can be corrected by IL-2 and suggest that Neurotropin restores immunoregulatory activity in B/W F1 mice by the recovery of IL-2 production.

Adjuvants, Immunologic↗
PubMed1989

Cathepsin B and L activities in gastric cancer tissue: correlation with histological findings.

Cathepsin B and L activities in cancerous and noncancerous mucosal tissues from 29 patients with gastric cancer were determined with a small amount of tissue homogenate. Both enzyme activities were significantly higher in cancerous tissues than in noncancerous tissues. The cathepsin B activity was higher with decreasing differentiation of the cancerous tissues, and also with increasing depth of invasion and metastasis to regional lymph nodes. Significantly high cathepsin B activity was observed in specimens of poorly differentiated adenocarcinomas, as well as in specimens from patients with extensive metastasis to n2 or n3 lymph nodes. These results suggest that high cathepsin B activity is characteristic of gastric cancer which invades and metastasizes. Therefore, in cases of marked elevation of cathepsin B activity in cancerous tissues, relatively extensive resection may be necessary to obtain a cure.

Adenocarcinoma↗
PubMed1989

Characterization of specific high-affinity receptor for human lymphotoxin.

The specific cell surface receptors for lymphotoxin (LT) which are expressed on murine fibroblast L.P3 cells, a subline of L929 cells, were found to consist of a single class of specific high-affinity receptors with a dissociation constant (Kd) of 3.8 X 10(-10) M and a density of 5.8 X 10(3) sites/cell. Similarly, murine fibroblast L929 cells, human melanoma A375 cells and human cervical carcinoma HeLa-S3 cells had about 7.2 X 10(3), 3.5 X 10(3), and 6.6 X 10(3) sites/cell with Kd values of 1.4 X 10(-10), 0.5 X 10(-10), and 1.1 X 10(-10) M, respectively. Among the LT receptor-positive cell lines, there was no direct correlation between the level of specific LT binding and the sensitivity to the cytotoxic or cytostatic effect of LT. Cross-linking of 125I-LT to the cell surface receptors with disuccinimidyl suberate, followed by two-dimensional gel electrophoresis of the cell lysate, revealed two kinds of LT-LT receptor complexes with molecular weights of 70 and 97 kDa, and having the same pI value of 6.8. Cell-bound 125I-LT was internalized within 1 h and degraded intracellularly, and finally secreted into the medium within a few hours. Appropriate concentrations of LT and interferon gamma (IFN gamma) showed synergistic cytotoxicity toward murine fibroblast L.P3 cells and human monocytoma U937 cells, but these cytokines were only slightly cytotoxic individually. Preincubation of these cells with IFN gamma increased the total number of LT receptors without any significant change in the dissociation constant or in the molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Concanavalin A receptor(s) possibly interacts with at least two kinds of GTP-binding proteins in murine thymocytes.

A part of the GTP gamma S-binding activity in murine thymocyte membranes was found to have affinity to a concanavalin A (Con A)-Sepharose column. The material was identified as Gi (inhibitory GTP-binding protein) on the basis of the molecular weight and by islet activating protein-dependent ADP-ribosylation and anti-alpha i (alpha subunit of Gi) immunoblotting. However, when the membranes prepared from Con A-stimulated thymocytes were used, no GTP gamma S-binding activity was detected in the Con A-bound fraction, suggesting that Gi physically and specifically associated with Con A acceptors dissociates upon Con A stimulation. Furthermore, another GTP gamma S-binding protein (25 kDa), which is quite similar to a novel phosphoinositide-specific phospholipase C (PI-PLC)-associated G protein in calf thymocytes (Wang, P., Toyoshima, S., & Osawa, T. (1988) J. Biochem. 103, 137-142), was detected among the Con A-Sepharose-bound proteins with the chemical cross-linking technique. When the 40 kDa and 25 kDa G proteins associated with Con A receptor(s) were isolated and their direct effects on the activity of partially purified PI-PLC as to phosphatidylinositol 4,5-bisphosphate hydrolysis were examined, the 25 kDa G protein was found to enhance the PI-PLC activity more effectively. On the other hand, pretreatment of cells with islet-activating protein completely abolished the inhibitory effect of Con A on the prostaglandin E1 and isoproterenol-induced increases of cellular cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Purification and partial amino acid sequences of a phospholipase C-associated GTP-binding protein from calf thymocytes.

We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987) J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP gamma S, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP gamma S-binding form and assayed as to the radioactivity of the [35S]GTP gamma S-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4,5-bisphosphate hydrolysis. From approximately 5.6 g of membrane protein we obtained about 5 micrograms of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.

Amino Acid Sequence↗
PubMed1989

Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity.

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.

Animals↗
PubMed1989

Alpha 2,3-linked sialic acids are more abundant in CD45 antigens and leukosialins of abnormal T cells of lpr mice than in those of normal T cells.

T cells from enlarged lymph nodes of MRL/MpJ-lpr/lpr (lpr) mice were found to express more binding sites for strongly hemagglutinating Phaseolus vulgaris agglutinin (PHA-E4) and fewer binding sites for Ricinus communis aglutinin (RCA) than those from normal MRL/MpJ-+/+ (+/+) mouse lymph node. We found that high-molecular-weight (180K-220K) glycoproteins on lpr T cells were strongly stained with these lectins on Western-blotting. These glycoproteins were found to belong to the CD45 family, by absorption with monoclonal anti-CD45 antibody. We also found that the other glycoproteins (105K and 120K glycoproteins on lpr T cells and a 105K glycoprotein on +/+ T cells) were strongly stained with the lectins which preferentially bind to mucin-type (O-linked) sugar chains on the cell surface. These glycoproteins were found to be leukosialins, by absorption with anti-leukosialin serum. From the results of the lectin-binding to these glycoproteins after sialidase treatment, CD45 antigens and leukosialin molecules on lpr T cells were found to have many more terminal alpha 2,3-linked sialic acids than those on +/+ T cells, and this fact explains why lpr T cells have more binding sites for PHA-E4 but fewer binding sites for RCA.

Animals↗
PubMed1989

Two populations of mouse lymphokine-activated killer cells separated by use of soybean agglutinin.

We separated lymphokine-activated killer (LAK) cells induced from spleen cells of BALB/c mice by culturing with recombinant interleukin-2 (rIL-2) into soybean agglutinin-positive (SBA+) and soybean agglutinin-negative (SBA-) fractions with a cell sorter at the time when LAK activity reached a maximum (day 3). We found that the cells with LAK activity were enriched in the SBA+ fraction. Analysis of cell surface phenotypes revealed that the SBA+ cells are of non-T cell origin, while the SBA- fraction consists of T cells. We also found that the large granular lymphocyte (LGL) fraction of spleen cells obtained with a Percoll gradient became SBA+ after culture for 3 days with rIL-2, whereas cells of high density did not. The change in SBA binding sites was also examined on C57BL/6 mouse spleen cells and we found that NK1.1+ non-T cells selectively acquire SBA binding sites at an early stage of activation with rIL-2. On the other hand, sorted SBA- cells gradually acquired SBA binding sites on extended culturing with rIL-2. These results suggest that the expression of SBA binding sites is related to the stage of cell activation with rIL-2, though cells of the NK-lineage became SBA+ at an earlier period of a culture with rIL-2 than cells of the T-lineage. Utilizing these findings, we separated NK-derived LAK cells by means of SBA-binding, and used the separated cells for adoptive immunotherapy for experimental pulmonary metastasis. We found that SBA(+)-NK cell-derived LAK cells showed stronger activity for the inhibition of experimental pulmonary metastasis than SBA(-)-T cell-derived LAK cells.

Animals↗
PubMed1989

Monoclonal antibodies against human platelet membrane glycoprotein IIIa and collagen-induced platelet activation.

Two monoclonal antibodies against human platelet membrane glycoprotein IIIa (GPIIIa) were obtained. One monoclonal antibody, designated as 1B1, was found to inhibit both collagen-induced platelet aggregation and release reactions. This antibody also inhibited the binding of 125I-labeled collagen to human platelets. On the other hand, the other antibody, designated as B10, had no effect on platelet activation induced by a number of physiological stimulants including collagen. Direct binding studies involving 125I-labeled 1B1 or B10 demonstrated that the binding sites for these antibodies on unstimulated platelets have dissociation constants of 4.2 and 14.0 nM, respectively. The binding of 125I-labeled 1B1 or B10 to platelets was not inhibited by the other antibody. Purified 1B1 and B10 were covalently coupled to Affi-Gel and then proteolytic fragments of GPIIIa were applied to the Affi-Gel immunoadsorbent columns. Of the several proteolytic fragments, the 56 kilodaltons (kDa) fragment obtained on digestion with V8 protease bound to both of the columns. The 69 and 55 kDa fragments obtained with BrCN bound to only the 1B1 Affi-Gel column, while the 63 kDa fragment obtained with chymotrypsin only bound to the B10-Affi-Gel column. Based on the partial amino acid sequences of these fragments and the amino acid sequence of GPIIIa (C. A. Fitzgerald, B. Steiner, S. C. Rall, Jr., S. Lo and D. R. Phillips, J. Biol. Chem., 262, 3936 (1987), the epitopes for 1B1 and B10 were concluded to be located at amino acids 335 to 582 and 206 to 335, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗